Comparative analysis of recombinant trypsin variants for enhanced MS-based peptide identification and quantification

Introduction: Trypsin is the most popular protease in proteomics for the digestion of proteins into smaller peptides, making them ideal for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. One of the most used Trypsin proteases in proteomics is the native Trypsin purified from pig pancreas. However, native Trypsin is prone to autolysis, leading to the formation of pseudotrypsin, which can interfere with peptide spectra analysis. To mitigate this, native Trypsin is modified by reductive methylation of lysine residues, enhancing its activity by preventing autolytic digestion. In addition, native Trypsin is further treated with TPCK to inhibit possible chymotrypsin contamination activity. Due to these extensive modifications, the purification of Trypsin from porcine pancreas remains challenging. Recombinant Trypsin produced by genetic engineering offers high purity and batch-to-batch consistency, presenting distinct advantages in proteomics studies, such as the absence of contaminating chymotrypsin, increased stability, minimized autolysis, and enhanced reproducibility across experiments.

In this study, we compared the performance of commercial Trypsin with recombinant Trypsins from Sus scrofa and Streptomyces griseus, both produced in the yeast Pichia pastoris. We evaluated digestion efficiencies, missed cleavage rates, and substrate efficiency at different time points and conditions.

Methods: Codon-optimized sequences for S. scrofa and S. griseus Trypsin genes were synthesized and inserted into Pichia pastoris. Induced under controlled fermentation conditions, recombinant Trypsins were harvested and purified. Protein extracts from HeLa cell line underwent a robust proteomics sample preparation employing the Protein Aggregation Protocol (PAC) protocol. Recombinant and commercial sequencing-grade Trypsins were compared for protein digestion under different digestion conditions. Mass spectrometric analysis was performed using an EvoSep One liquid chromatography system coupled to an Astral mass spectrometer operated in data-dependent acquisition (DDA) mode. MaxQuant software suite was utilized for peptide identification and quantification prior to further downstream data processing using R.

Results: Recombinant Sus scrofa Trypsin demonstrated comparable performance and efficiency at digesting cellular extracts as commercial Trypsin, with no significant impact from the absence of lysine methylation, even during extended digestion times (24 hours). Recombinant Streptomyces griseus trypsin exhibited good cleavage efficiency but identified fewer peptides than porcine Trypsins. Additionally, we evaluated the performance of recombinant trypsin in combination with LysC, another commonly used enzyme in proteomics. Our results show that the digestion efficiency improves simultaneously as the miss cleavages decrease when both enzymes are combined.